4.3. Preparation of Tissue Homogenates

The frozen liver was cut in smaller pieces with a sharp scalpel. Afterwards, the tissue pieces (about 1 g wet weight in total) were transferred to a stainless steel mortar and immediately doused with liquid nitrogen. The frozen pieces were ground with a pestle upon reaching a homogenous powder-like state (~5 min). During this procedure, liquid nitrogen was continuously added to avoid tissue thawing. The powder was aliquoted and weighed to determine the wet weight. Different solvents H₂O, H₂O/MeOH = 1/1 (v/v), H₂O/MeOH = 1/1 +1% SDS, or MeOH were added to suspend at the respective concentration. The homogenate was vortexed at 3200 rpm for 10 s and another 10 s prior to sample taking.

A small piece, of approximately 20–70 mg, was cut off a frozen liver and transferred in a Precellys cup with ceramic beads (V = 2 mL). Different solvents H₂O, H₂O/MeOH = 1/1 (v/v), H₂O/MeOH = 1/1 +1% SDS, or MeOH were added to suspend the samples at the concentration of 0.05 mg/µL. The sample was directly homogenized in the previously added solvent with a Precellys^® 24 tissue homogenizer from Bertin Instruments (Berlin, Germany). The homogenizer was operated at 5000 rpm, two cycles of 15 s run time, and a 60 s break interval between both cycles.